1. Field of the Invention
The invention is directed to a laser scanning microscope with at least one light source emitting illumination light for illuminating a specimen, at least a first detector for detecting the detection light proceeding from the specimen, and an objective through which the specimen can be illuminated and detected, the objective being arranged in an illumination beam path and in a detection beam path, and with an additional non-descanned detection beam path, particularly for detecting detection light originating from nonlinear excitation by means of pulsed laser light.
2. Description of Related Art
A microscope of the type mentioned above is described, for example, in DE 19702753 and is shown by way of example in FIG. 1 as prior art.
Non-descanned detectors are provided in an out-coupling between the objective and tube lens and on the side of the specimen located opposite the objective for transmission.
Non-descanned detection (NDD) has established itself as a standard method for nonlinear laser scanning microscopy. In this detection mode, the light emitted from the specimen is detected integrally by means of a large-area sensor rather than by the scanning optics or through a pinhole. Focusing optics can be provided in front of the detector.
A small specimen volume can be excited and imaged through the nonlinear processes during excitation also without confocal detection. Further, this integral detection mode uses a relatively large portion of the light emitted by the specimen.
The detection efficiency can be further increased when the NDD is situated as near to the microscope objective as possible. This was achieved by means of a lateral out-coupling in the vicinity of the objective exit pupil.
Arrangements of this type are also described in U.S. Pat. No. 6,437,913 B1 and DE 101 20 424 B4.
Since NDDs are used primarily in electrophysiological applications and in apparatus with objective turret focusing, the known type of out-coupling is difficult. One reason for this is that the out-coupling unit and the NDD assembly must be moved along with focusing; another reason is that the detectors which protrude at the sides limit the accessibility to specimens that is required for electrophysiological applications (manipulation, patch clamp). This additional weight load, which is also asymmetrical primarily because of the lateral out-coupling, dramatically reduces the positioning accuracy and the traveling speed of the focusing unit. An added difficulty arises in that it is also becoming increasingly important for applications using biological specimens that a plurality of wavelength regions be detected independently from one another. As a result of this, two or more NDD units are cascaded, which increases the moved mass twofold or more and this mass acts by way of a lever arm that increases in size in proportion to the cascading.